Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Germany.
Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany.
Methods. 2019 Mar 1;156:102-109. doi: 10.1016/j.ymeth.2018.10.020. Epub 2018 Oct 28.
In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (mA). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using mA-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.
在 RNA 修饰这一不断发展的领域中，使用抗体的沉淀技术发挥着重要作用。然而，人们对这些抗体的特异性知之甚少，也缺乏评估其有效性的方案。在这里，我们提出了一种方法来评估 MeRIP 型下拉实验后的富集因子，此处用针对 N6-甲基腺苷（m6A）的商业抗体进行了示例。通过测试不同的下拉和洗脱条件，我们使用含有 m6A 的 mRNAs 针对相同序列的未修饰对照物测量了 4-5 的富集因子。这两种类型的 mRNA 在不同的核苷酸处都带有 P 标记，允许在消化成核苷酸、TLC 分离以及标记物的定量磷显影后，在混合物中对它们进行相对定量。
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